Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.     

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.     

Acute dosing of latrepirdine (Dimebon™), a possible Alzheimer therapeutic, elevates extracellular amyloid-β levels in vitro and in vivo

Background

It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm²) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined.

Results

The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.

Conclusion

The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.     

Effects of mitochondria-selective fluorescent probes on mitochondrial movement in Arabidopsis mesophyll cells evaluated by using the quantification

Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.     

Docosahexaenoic acid accumulation in the prenatal brain: a purported role in combating oxidative stress

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.     

Neural Coding of Finger and Wrist Movements

Previous work (Schieber and Hibbard, 1993) has shown that single motor cortical neurons do not discharge specifically for a particular flexion-extension finger movement but instead are active with movements of different fingers. In addition, neuronal populations active with movements of different fingers overlap extensively in their spatial locations in the motor cortex. These data suggested that control of any finger movement utilizes a distributed population of neurons. In this study we applied the neuronal population vector analysis (Georgopoulos et al., 1983) to these same data to determine (1) whether single cells are tuned in an abstract, three-dimensional (3D) instructed finger and wrist movement space with hand-like geometry and (2) whether the neuronal population encodes specific finger movements. We found that the activity of 132/176 (75%) motor cortical neurons related to finger movements was indeed tuned in this space. Moreover, the population vector computed in this space predicted well the instructed finger movement. Thus, although single neurons may be related to several disparate finger movements, and neurons related to different finger movements are intermingled throughout the hand area of the motor cortex, the neuronal population activity does specify particular finger movements.     

AMP‐activated protein kinase mediates activity‐dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 557–573, 2014     

Interaction between mitochondria and oxidative stress in neuronal injury

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.     

Mitochondria are morphologically and functionally heterogeneous within cells

We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility

The fasciculation and elongation protein zeta-1 (FEZ1), a mammalian orthologue of Caenorhabditis elegans UNC-76 protein, is a 45-kDa protein with four coiled-coiled domains and efficiently promotes the neurite elongation in the rat phaeochromocytoma PC12 cells. UNC-76 proteins of C. elegans and Drosophila have been genetically demonstrated to be involved in the axonal guidance. We here show that FEZ1 RNA interference (RNAi) represses the formation of axon in rat embryo hippocampal neurons. An anterograde mitochondrial movement is also retarded in neurites of the RNAi-treated hippocampal neurons. Moreover, the size of mitochondria is considerably elongated by the RNAi treatment. The transport of mitochondria from soma to axon or dendrites is essential for the neuronal differentiation. Therefore, our results strongly suggest that FEZ1 participates in the establishment of neuronal polarity by controlling the mitochondrial motility along axon.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Regulation of mitochondrial morphology and inheritance by Mdm10p,a protein of the mitochondrial outer membrane

Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.     

The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.     

Live cell imaging of mitochondrial movement along actin cables in budding yeast

BACKGROUND: Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.     

A dynamical model of the transport of asbestos fibres in the human body

We present a model for the transport of a single type of asbestos fibre through the human body. The model captures the transport modes that pertain particularly to the lungs and the mesothelium. Numerical solutions of the system follow observed movement in the body. We compare the accumulation of fibres in the lungs versus the mesothelium, and then we give analysis and results for various cases of exposure level and exposure time. Models, such as the one developed here, can give clues as to how asbestos fibres impact the body, and where to look for major impact.     

Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons

Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

Specificity of sensorimotor learning and the neural code: Neuronal representations in the primary motor cortex

Human studies show that the learning of a new sensorimotor mapping that requires adaptation to directional errors is local and generalizes poorly to untrained directions. We trained monkeys to learn new visuomotor rotations for only one target in space and recorded neuronal activity in the primary motor cortex before, during and after learning. Similar to humans, the monkeys showed poor transfer of learning to other directions, as observed by behavioral aftereffects for untrained directions. To test for internal representations underlying these changes, we compared two features of neuronal activity before and after learning: changes in firing rates and changes in information content. Specific elevations of firing rate were only observed in a subpopulation of cells in the motor cortex with directional properties corresponding to the locally learned rotation; namely cells only showed plasticity if their preferred direction was near the training one. We applied measures from information theory to probe for learning-related changes in the neuronal code. Single cells conveyed more information about the direction of movement and this specific improvement in encoding was correlated with an increase in the slope of the neurons' tuning curve. Further, the improved information after learning enabled a more accurate reconstruction of movement direction from neuronal populations. Our findings suggest a neural mechanism for the confined generalization of a newly acquired internal model by showing a tight relationship between the locality of learning and the properties of neurons. They also provide direct evidence for improvement in the neural code as a result of learning.